Immunological reactions are highly specific interactions in which an antigen binds with a corresponding constituent specific to the antigen and generally known as the antibody, to form an immunological complex. In a biological system, the entry of a foreign biological constituent causes the biological system to produce the specific antibody to the antigen. The antibody molecules have chemical binding sites which compliment those on the antigen molecules so that the antigen and the antibody bond to form the immunological complex.
Antibodies are produced by biological systems in response to invasion thereof of foreign bodies. Even the antibody of one system can act as an antigen in another system and invoke an immunological reaction.
Consequently, the detection of either antigens or antibodies present in a biological system is of medical diagnostic value in determining which antigens are present or to which antigens the system has been exposed.
Most antigens are proteins or contain proteins as an essential part, whereas all antibodies are proteins. An antigen and an antibody protein may each have several binding sites.
Additional immunological reactions other than antigen-antibody reactions are also contemplated by the invention. For instance, the following systems include biological particles which are capable of undergoing the immunological reactions described herein:
Viruses PA1 Bacteria and Bacterial Toxins PA1 Fungi PA1 Parasites PA1 Animal Tissue PA1 Animal Body Fluids and the like.
An illustration of antigens would be goat antibody, human chorionic gonadotrophin (HCG) and hepatitis associated antigen (HAA).
Antigens, with respect to viruses, are viral particles, and the corresponding antibody is produced by administration of the antigen to a living host. Illustrations of such antigen-antibody complexes useful in the herein disclosed invention are: Rubella virus antigens--Rubella virus antibody; polio virus antigen--polio virus antibody; versicular stomatitis virus (VSV) antigen--VSV antibody, and acquired immune deficiency syndrome (AIDS) antigen--AIDS antibody.
Antibodies corresponding to bacteria and bacterial toxins are produced in a manner similar to virus antibodies. The following are illustrative examples of bacteria or bacteria toxin antigen--antibody pairs which can be used in the present invention: tetanus toxoid suspension (antigen)--tetanus antibody; diphtheria toxin suspension (antigen)--diphtheria antibody; Neisseria genorrhoea suspension (antigen)--gonorrhoea antibody; Treponema palladium suspension (antigen)--syphilis antibody.
Fungi antigens are antigenic extracts of fungal suspensions, and the antibody is the fungal antibody produced by introduction into a living host. Illustrations of fungi system antigen-antibody complexes are: Aspergillus extract suspension (antigen)--aspergillus fungus antibody; Candida extract suspension (antigen)--candida fungus antibody.
Antigens and antibodies in parasite systems are tested in a similar manner. The system Toxoplasma gondii extract (antigen)--Toxoplasma gondii antibody is exemplary.
Polysaccharide is a system wherein the antigen is a carbohydrate antigen such as the antigen--antibody system of pneumococcus polysaccharides (antigen)--pneumococcus antibody.
The antigenic constituent with respect to hormones is usually found in a hormone extract, and the antibody is the particular hormone antibody elaborated by the living organism after injection. An illustration of this antigen-antibody complex is insulin-hormone-insulin antibody.
Specific antigens or antibodies can also be labeled with an enzyme. Typically, in an enzyme-linked surface-immuno-assay [ELISA], the antigen is adsorbed to a surface in a little well. The sample suspected of containing antibody is added to the well and allowed to incubate. If the sample contained antibody, some of the antibody will have specifically attached to the preadsorbed antigen. The well is then rinsed and a second antibody linked to an enzyme is added to the well. This second antibody may be specific for either the antigen or for the first antibody but not for both. If the second antibody is specific for the antigen, this is called a competition assay. If the second antibody is specific for the antibody, this is called a sandwich assay. The well is again rinsed, and the proper substrate for the enzyme is added to the well. The enzyme, if present, will react with the substrate, typically in such a manner that the color of the solution changes. The change in color is accordingly correlative to the amount of antibody in the first solution.
Certain other enzymes will cause a precipitate to form, and the detection of, the presence of, and the amount of that precipitate will indicate the presence of and the concentration of the antibody suspected in first solution.
Arwin et al., U.S. Pat. No. 4,072,576 studied enzymatic reactions by measuring the electrical potential difference over membranes specific for the reaction product of the reaction between an enzyme and a specific substrate for that enzyme. Such a method is not broadly applicable to a wide variety of immunologically-active substances.
In Giaever, U.S. Pat. No. 4,054,646, such immunological reactions are shown to take place on a metal globule-coated substrate. The resultant antigen-antibody complex can then be examined by optical means, and by contrasting the thickness of the bare antigen or antibody with the thickness of the antigen-antibody complex. In McConnell, U.S. Pat. No. 4,490,216, such antigen-antibody complexes may be detected by changes in electrostatic interaction between a polarity layer and an amphiphilic layer linked to the antigen-antibody complex and separated from the polarity-sensitive layer by a lipid layer. In Janata, U.S. Pat. No. 4,151,049, detection of the presence of chemical substances is accomplished by measuring the change in electrical charge of an electrode encased with a membrane comprising a hydrophobic organic polymeric substrate with hydrocarbon chains with specific proteins (antigen or antibodies) adsorbed thereon. A long time--of the order of one to two days--is required to complete the measurement.
It is the object of the present invention to detect, more quantitatively, and by electrical means, the immune reaction by use of enzyme-linked immunologically-active substances and the enzyme-substrate reaction.
A primary advantage of the present invention is the simplicity of the electrical measurements employed. A further advantage is that the procedure can be carried out rapidly. In another advantageous embodiment, several electrodes may be used in a single liquid medium to be analyzed for the presence of several immunologically-active substances. This permits many different immunologically-active substances to be analyzed at the same time.